Detection of co-harboring OXA-58 and NDM-1 carbapenemase producing genes resided on a same plasmid from an Acinetobacter pittii clinical isolate in China

Objective(s): Acinetobacter pittii has become an emerging opportunistic noscomial pathogen worldwide with multi-drug resistance. In the present study, an A. pittii strain was isolated from bronchoalveolar lavage fluid sample harboring both OXA-58 and NDM-1carbapenemase producing genes. The mechanisms of carbapenem resistance of the A. pittii strain was investigated. Materials and Methods: Carbapenemase producing genes were examined by PCR and DNA sequencing. S1-PFGE was used to localize carbapenemase encoding genes. Filter mating and electrotransformation were used to investigate the transferability of such carbapenemase encoding genes between different strains. Genetic surroundings of blaOXA-58 and blaNDM-1 genes were detected as well. Results: The A. pittii strain, carrying both OXA-58 and NDM-1 carbapenemase encoding genes, was resistant to all β-lactam antibiotics, while suscepitible to ciprofloxacin, levofloxacin, tobramycin, cotrimoxazole and tigecycline. Southern blot hybridization for the blaOXA-58 and blaNDM-1 gene indicated that the two genes locate in the same plasmid with molecular weight of 310.1-336.5kb. BlaOXA-58 was located in an ISAba3-blaOXA-58-ISAba3-like structure, and the blaNDM-1 gene cluster was embedded in an ISAba125-aphA6- blaNDM-1-bleMBL-ΔtrpF-dsbC-cutA structure sequentially. Conclusion: In the present study, it is first reported an A. pittii clincal strain in China, co-harboring OXA-58 and NDM-1 carbapenemase producing genes residing on a same plasmid. In hospital and community settings, it is of great significance and urgence to increase the surveillance of these kinds of organisms.


Introduction
Acinetobacter spp. are frequent pathogens responsible for nosocomial infections, including Acinetobacter baumannii as the predominant species followed by Acinetobacter pittii and Acinetobacter nosocomialis (1). Since the phenomenon of carbapenem resistance increasingly emergences, there is a big challenge of multidrug resistant Acinetobacter in treating hospital infections (2,3).
To our knowledge, the increasing expression of OXA type carbapenemase genes is the most frequent mechanism of carbapenem resistance in Acinetobacter. It mainly includes the intrinsic bla OXA-51 -like gene as well as the horizontally acquired genes such as bla OXA-23 -like, bla OXA-24 -like and bla OXA-58 -like genes (4,5). The bla OXA- 23 -like genes are much more prevalent than the bla OXA-24 -like and bla OXA-58 -like genes. All of them are capable of yielding carbapenem resistance in a high level and result in serious local outbreaks (6)(7)(8). Moreover, the expression of metallo-β-lactam (MBL) carbapenemase also plays an important role in carbapenem resistance of Acinetobacter, including IMP, VIM, SIM, and NDM detected previously in Acinetobacter (9). Although these MBLs genes were less detected than OXAs, their carbapenemase activities were typically much higher (10,11). Especially, since 2008, there have been an increasing number of reports about the dissemination of NDM-1-producing Acinetobacter spp in many countries and it resulted in a major threat for clinical treatments in view of its highly frequent co-occurrence with other resistance genes (12)(13)(14). Recently, the global spread of blaNDM-1-harboring A. pittii strains is fierce. With these powerful genes, as the reservoirs for dissemination, they are able to transfer highly across various bacterial species (15)(16)(17).
In this study, we investigated the antibiotic susceptibility, genetic environment, and transferability of a single clinical A. pittii isolate co-harboring blaNDM-1 and blaOXA-58 genes on a same plasmid, in order to improve awareness of the urgency of carbapenemaseproducing A. pittii isolates in China.

Bacterial isolates
The A. pittii was isolated from a bronchoalveolar lavage fluid sample of a 56-year-old man suffering from chronic obstructive pulmonary disease (COPD). It was initially identified as Acinetobacter calcoaceticus baumannii by Vitek 2 system (bioMérieux, Marcy l'Etoile, France). To confirm the identity of this strain, a fragment of the 16S rRNA gene was amplified using primers 27-forward (5'-AGA GTT TGA TCC TGG CTC Carbapenemase in Acinetobacter pittii Chen et al. AG -3') and 1492-reverse (5'-GGT TAC CTT GTT ACG ACT T -3') by PCR [10] and the resultant PCR product sequenced as A. pittii with 100% identity, designated as AB34.

Antimicrobial susceptibility testing
The minimum inhibitory concentration (MIC) of various antibiotics was detected on the Vitek 2 system (bioMérieux, Marcy l'Etoile, France).

S1-PFGE and southern blot
The total bacterial DNA was first prepared in agarose plugs, digested with S1 nuclease (Takara, Japan) and further separated by PFGE, as reported previously (21). The DNA fragments were transferred horizontally to a nylon membrane (Millipore, USA), hybridized with digoxigen in-labeled bla OXA-58 and bla NDM-1 probe and detected using a nitroblue tetrazolium/5-bromo-4chloro-3'-indolyl-phosphate color detection kit (Roche Applied Sciences, USA).

Filter mating experiment
Filter mating experiment was performed with the rifampin-resistant EC600 and azide-resistant E. coli J53 as the recipient strains. The transconjugants were selected on Mueller-Hinton agar plates containing [ampicillin (50 mg/l) and rifampicin (1024 mg/l)] or [ampicillin (50 mg/l) and NaN 3 (200 mg/l)], respectively, and incubated for 16-18 hr at 37 °C . The successful transconjugants were selected on Mueller-Hinton agar incorporating the same concentration of antibiotics mentioned above. The transformants would be confirmed the resistant genes by PCR.

Plasmid construction and electrotransformation
The plasmid DNA of isolate AB34 was extracted, digested by restriction enzyme EcoRI or SacI and then cloned into the cloning vector pPet28a. The conjugant OXA-23-live was electrotransformed to E. coli DH5α competent cells and selected on Mueller-Hinton agar plates containing ampicillin (50 mg/L) in order to obtain the E. coli clone expressing the corresponding carbapenemase enzyme.

Genetic surroundings detection
A total genomic sample of A. pittii strain AB34 was extracted and purified using the Wizard Genomic DNA purification kit (Promega Corporation, Madison, WI) according to the manufacturer's instructions. DNA concentration was estimated using a Qubit dsDNA HS Assay Kit and a Qubit 3.0 Fluorometer (Invitrogen, Thermo Fisher Scientific, USA). Extracted DNA was then sequenced with a standard 2×125 paired-end runs protocol on an Illumina HiSeq 2000 (Illumina, San Diego, CA, USA). The quality of the high-throughput sequence data was assessed by FastQC (http://www. bioinformatics.babraham.ac.uk/projects/fastqc/). Raw sequence reads were then de novo assembled using Plasmid SPAdes 3.9.0 (http://bioinf.spbau.ru/ spades)(22), in order to identify plasmid contigs, and the quality was assessed by QUAST (http://quast.bioinf. spbau.ru).

Nucleotide sequence accession numbers
The bla OXA-58 or bla NDM-1 nucleotide sequences are available in GenBank under the accession number KF208466 or KF208467, respectively.

Susceptibility results
The isolate AB34 was resistant to all β-lactams including ceftazidime, cefepime and carbapenems as well as ampicillin/sulbactam inhibitor combinations, but remained susceptible to cotrimoxazole, tobramycin, ciprofloxacin, levofloxacin and tigecycline ( Table 2).

Detection of carbapenemases encoding genes
Only OXA-58(599bp) and NDM-1(720bp) were detected in this strain. The amplified products were confirmed by sequencing. BLAST version 2.2.24 (http://blast.ddbj. nig.ac.jp/) was used to process the sequencing data and identify genes.

Location of bla OXA-58 and bla NDM-1 genes
After PFGE ( Figure 1) and southern blot hybridization (Figure 2), it was found that both bla OXA-58 and bla NDM-1 genes resided on a same 310.1-336.5kb plasmid. Horizontal transfer of the two carbapenem resistance determinants from AB34 to EC600 or E. coli J53 was not detected in filter mating.

Genetic surroundings
A BLAST search against all completely sequenced blaOXA-58 and blaNDM-1-genes-co-harboring plasmids in GenBank (http://www.ncbi.nlm.nih.gov/GenBank/)  showed that the blaOXA-58 gene in AB34 was located down-stream of the shortened ISAba3 gene, and upstream of a complete copy of ISAba3 gene (Figure 3).

Discussion
Since the last decade, carbapenemase-producing Acinetobacter spp have disseminated rapidly throughout the world, posing an urgent threat to public health (1,23). A. pittii, formerly named Acinetobacter genomic species 3, is increasingly recognized as a clinically important pathogen within the Acinetobacter calcoaceticus-A. baumannii complex, which addresses a particular concern due to its competency to acquire multidrug resistance against a wide range of antimicrobial agents (24).
The first known OXA-58-producing Acinetobacter strain was isolated in France in 2003 (9). It shares less than 50% of amino-acid homology with oxacillinase. OXA-58 is a widely spread carbapenem-hydrolyzing class D β-lactamases (CHDLs) that has been reported in Acinetobacter spp. from Europe (25), Argentina (26), Australia (27), the United States (28) and many Asian countries (29). Though OXA-58 shows only low carbapenem-hydrolyzing activity in vitro, the insertion sequence upstream of bla OXA-58 enhances its transcription greatly and mediates resistance to carbapenems (30)(31)(32). It is speculated that the insertion of other IS element into ISAba3-like could generate a hybrid promoter to enhance the transcription of bla OXA-58 and mediate greater carbapenem resistance than the intact ISAba3like element as previously reported (32,33). However, in China, the most common carbapenamase-producing type of A. baumannii is OXA-23,while OXA -58 is rarely reported (34).
The NDM-1 gene encodes an enzyme that hydrolyses and inactivates all β-lactam antibiotics including carbapenems, except for aztreonam, and thus induces resistance to carbapenems (35). A. baumannii carrying NDM-1 have been reported from clinical and environmental isolates in several countries (36)(37)(38)(39). Not only Acinetobacter spp. act as reservoirs for blaNDM genes in non-human settings, as recently shown in several Chinese studies with identification of NDM-1producers among A. calcoaceticus and Acinetobacter junii from environmental samples from livestock farms (40), Acinetobacter johnsonii from hospital sewage (40) and Acinetobacter lwoffii from chickens (40), but also act as a source of bla NDM genes then horizontally transferred to enterobacterial species as evidenced (41).
It is noteworthy that coexistence of blaNDM and blaOXA has been described in Acinetobacter, e.g. blaOXA-23 and blaNDM-1 in A. baumannii from India (42) and the Czech Republic (43), and bla NDM-1 , bla  and bla IMP in A. baumannii from China (44). However, it remains unclear whether and how these co-existing carbapenemase genes are expressed to contribute to drug resistance.
A. pittii 44551 was recovered from a patient with gout combined with tuberculosis and was found to harbor the carbapenemase genes bla NDM-1 and bla OXA-58 on two different plasmids pNDM-44551 and pOXA58-44551, respectively, from China in 2015 (1). Emergence of ST119 A. pittii AP 882 co-harbouring NDM-1 and OXA-58 in Malaysia was reported as well, of which genes encoding NDM-1 and OXA-58 resided on an ca.140 kb mega plasmid and a 35 kb plasmid, respectively (45).
Similarly, in our present study, AB34 was isolated and detected co-harbouring OXA-58 and NDM-1carbapenemase producing resided on the same 310.1-336.5kb plasmid. However, horizontal transfer of carbapenem resistance determinants from AB34 to EC600 or E. coli J53 (AzR) was not detected in filter mating experiment. The up-stream and down-stream of OXA-58 gene in AB34 are ISAba3, which shows 99% similarity to A. pittii pOXA-58-44551 (1). It is reported that the structure of OXA-58 of A. pittii 44551 is 372F-ISAba3-like-bla OXA-58 -ISAba3, where the bla OXA-58 contributed little to β-lactams resistance due to a lack of the blaOXA-58-driven promoter (1). An intact ISAba3like element upstream of bla  has been linked to a lower level of resistance to imipenem compared with bla OXA-58 with hybrid promoters such as IS6 family-ISAba3-like-bla OXA-58 .
The upstream of NDM-1 of AB34 is ISAba125, while the down-streams are arranged sequentially as aphA6, bla NDM-1 , bleMBL, ΔtrpF, dsbD, cutA1, GroES, UmuD, hypothetical protein, site-specific DNA methylaselike, ISAba31-like, dihydrofolate reductase putative membrane protein, C-5 cytosine-specific DNA methylase  family protein, ISAba14-like, which was with 99% sequence identity against that of Acinetobacter lwoffii pNDM-BJ01 from Beijing, China (46). It is proved that the genetic surroundings of bla NDM-1 is an important vector to mediate to integration and transfer. It should be noted that the ISAba125 element upstream of bla NDM-1 is usually intact in Acinetobacter but often truncated in Enterobacteriaceae, suggesting the probable spread of the bla NDM-1 genetic platforms from Acinetobacter to Enterobacteriaceae (47)(48)(49)(50).

Conclusion
This study has improved awareness of the urgence of carbapenemase-producing A. pittii isolates in China. Further investigations on the comparative genomic analysis of a large-scale sampling of A. pittii strains from a wide spatial and temporal range in the context of genomic epidemiological characteristics are currently on the way. These data highlight the molecular mechanisms contributing to the rapid development of antimicrobial resistance and will facilitate to expand our understanding of the global public health concern caused by Acinetobacter spp.